The goal of this program is to develop novel inhibitors of human palmitoyl acyltransferases (PATs) that are effective as cancer therapeutic agents. PATs represent new targets for anticancer drug development because of their pivotal roles in regulating the subcellular localization of specific oncoproteins. Most importantly, certain forms of ras-encoded proteins require palmitoylation for their targeting to the plasma membrane and for their ability to transform cells. Since post-translational processing of Ras proteins is critical for their function, the enzymes that catalyze these processing steps have been considered as potential targets for anticancer drugs. Inhibition of farnesylation has been the main focus of this therapeutic effort to date. However, the PATs may be even better targets, since palmitoylation is a dynamic process whereas farnesylation is not. A major reason that inhibition of palmitoylation has not yet been therapeutically exploited is that mammalian PATs have yet to be molecularly and biochemically characterized. Recent studies of palmitoylation in the yeast Saccharomyces cerevisiae have identified two putative PATs. Using the sequences of the yeast enzymes, we performed a search for homologues in vertebrates. Based on sequence and structural homologies, we selected a human protein called HIP3 for further study, i.e. to determine if this protein is an authentic human PAT (hPAT). Using RNA interference, we have shown that HIP3 has PAT activity, and that it affects the subcellular localization of palmitoylated proteins. In this SBIR program, these findings and our previously published work on hPATs will be used to develop small molecule inhibitors of hPATs to be evaluated as anticancer therapeutics. The following specific aims will be addressed in the Phase I project: 1. To identify compounds that inhibit hPAT activity by screening a diverse collection of small molecules in an established ass ay and a high-throughput yeast-based assay. 2. To use computational and medicinal chemistry to optimize these compounds. 3. To determine the in vitro activity, in vivo toxicity, pharmacokinetics and antitumor activity of lead PAT inhibitors.